Isolation and Expansion of Bone Marrow-Derived MSCs

Undifferentiated MSCs were derived from bone marrow aspirates obtained from a healthy adult female donor (25 years old) at the Hematopoietic Stem Cell Core Facility at Case Western Reserve University with informed consent under an Institutional Review Board-approved protocol. Briefly, a bone marrow sample was aspirated and washed with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. The sample was centrifuged at 460×g on a pre-formed Percoll density gradient to isolate the mononucleated cells. These cells were resuspended in serum-supplemented medium and seeded at a density of 1.8×10⁵ cells/cm² in 10 cm diameter plates. Non-adherent cells were removed after four days by changing the medium. The adherent cells were expanded in CCM at 37°C in a humidified atmosphere of 5% CO₂ (Valonen et al., 2010 (link)). The primary culture was trypsinized after approximately two weeks, cryopreserved using Freezing Medium (Invitrogen) for shipment and preparation of tissue engineered constructs.